Exosomes are small membrane-bound vesicles secreted by most cell types and play an important role in cell-to-cell communication. Increasing evidence shows that exosomal proteins in urine may be used as novel biomarkers for certain diseases. Purified urinary exosomes are necessary for downstream studies and application development. However, conventional methods for exosome isolation and enrichment are technically challenging and time-consuming. Poor specificity, low recovery and instrumental dependence also limit the use of these methods. It is particularly urgent to develop a rapid and efficient extraction method for basic research and clinical application. Particularly, urine is a dilute solution system with relatively low abundance of exosomes, due to which the isolation of urinary exosome requires more efficient technology. Here, we propose a new strategy for facile exosome isolation from human urine by utilizing the ultrafiltration technique and the specific interaction of TiO₂ with the phosphate groups on the lipid bilayer of exosomes. Downstream characterization and proteomic analysis indicate that high-quality exosomes can be obtained from human urine by this ultrafiltration–TiO₂ series method in 20 minutes, and 91.5% exosomes with an intact structure are captured from urine by this method. Moreover, 1874 protein groups have been identified through LC-MS. The results show that the protein identification of our method is 23% higher at least than those obtained by conventional strategies. We also identified 30 differential proteins by comparing the urinary exosomes from healthy male and female volunteers. These proteins are related to biological processes, such as lipid metabolism, fatty acid metabolism and nucleotide metabolism. Our analysis reveals that combining conventional ultrafiltration and TiO₂-based isolation is ideal to overcome the inherent limitations of identification of exosome proteins derived from urine, and yield highly pure exosome components for downstream proteomic analysis.